BR112015007480B1 - high efficiency sex based sperm separation methods. - Google Patents

Abstract

high efficiency sex based sperm separation methods. this disclosure relates to cell separation methods, and particularly cell separation methods that improve the separation efficiency or productivity in a particle separation instrument using a measured separation effectiveness parameter. in one embodiment, minimum productivity and minimum purity can be established and maintained while trying to maximize the effectiveness of the separation. while in another embodiment, minimum separation efficiency and minimum purity can be established and maintained while trying to maximize the productivity of a separation.

Description

(54) Title: METHODS OF SEPARATION OF SPERM BASED ON SEX OF HIGH EFFECTIVENESS.

(51) Int.CI .: A01N 1/02; C12N 5/076; C12N 5/071; 1/24 C12Q; C12Q 1/6806.

(52) CPC: A01N 1/02; C12N 5/061; C12N 5/0612; 1/24 C12Q; C12Q 1/6806.

(30) Unionist Priority: 10/05/2012 US 61 / 710,343.

(73) Holder (s): INGURAN, LLC.

(72) Inventor (s): KENNETH MICHAEL EVANS; THOMAS BOYD GILLIGAN; JOHNATHAN CHARLES SHARPE; JUAN MORENO; RAMAKRISHNAN VISHWANATH.

(86) PCT Application: PCT US2013028934 of 04/03/2013 (87) PCT Publication: WO 2014/055112 of 10/04/2014 (85) Date of the Beginning of the National Phase: 02/04/2015 (57) Summary: METHODS OF SEPARATION OF SPERM BASED ON SEX OF HIGH EFFECTIVENESS. This disclosure relates to cell separation methods, and particularly cell separation methods that improve the separation efficiency or productivity in a particle separation instrument using a measured separation effectiveness parameter. In one embodiment, minimum productivity and minimum purity can be established and maintained while trying to maximize the effectiveness of the separation. While in another embodiment, minimum separation efficiency and minimum purity can be established and maintained while trying to maximize the productivity of a separation.

/ 53

DESCRIPTIVE REPORT

Patent Application for "METHODS OF SEPARATING SPERM BASED ON SEX OF HIGH EFFECTIVENESS"

CROSS REFERENCE TO RELATED APPLICATION [001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 710,343 filed on October 5, 2012, the entire contents of which are incorporated herein by reference.

TECHNICAL AREA [002] Generally, this disclosure relates to cell separation methods, and it relates more particularly to separation methods that improve the efficiency and recovery of separate sperm based on sex.

BACKGROUND [003] The most widely used sperm separation methods are generally based on detecting quantifiable differences in the DNA content of X-chromosome-bearing sperm and Y-chromosome-bearing sperm. Several modifications to flow cytometers for this purpose have been described. in U.S. Patents 5,135,759, 6,263,745, 7,371,517 and 7,758,81 1, each of which is incorporated herein by reference. In many species, this difference in DNA content can be small. In cattle, for example, Holstein bulls have a difference of about 3.8% in DNA content, while Jersey bulls have a difference of about 4.1%. The inaccurate nature of stoichiometric DNA staining makes these small differences difficult to verify and requires exposure of sperm to harmful conditions over time.

[004 | Although Hoechst 33342 can be used in non-toxic concentrations, sperm must be incubated at elevated temperatures and pHs

2/53 raised for sufficient penetration of Hoechst 33342 with sufficient uniformity for analysis or separation. Each rise in sperm temperature and change in sperm pH can contribute to damage to the sperm. In addition, the pressure and shear forces applied to the sperm within a flow cytometer may additionally compromise the sperm membranes. These factors can accelerate the deterioration of sperm membranes, further reducing the already limited shelf life of viable sperm.

Accordingly, previous sperm separation efforts focused on using smaller insemination samples and producing the largest amount of separated sperm in the shortest amount of time. U.S. Patent 6,149,867, incorporated herein by reference, describes methods and devices aimed at helping sperm to better survive flow cytometric separation in combination with low-dose inseminates. Subsequent advances in flow separation have focused on improvements in detection or throughput. However, as speeds and productivity increased, larger amounts of sperm, including viable sperm of the desired sex, were discarded with waste. There are also additional trade-offs between purity and recovery. For example, where the desired purity is greater than 95%, less sperm can be determined with the required confidence level compared to purities of 70%, 80% or 90%, meaning that less sperm are separated at increasingly high purities and that more viable sperm are discarded with the waste stream.

[006] There are additional losses in efficacy with regard to discarded viable sperm due to the occurrence of events

3/53 coincident. A coincident event occurs when two or more sperm are too close together to be separated. In any event, all sperm can be discarded with waste, whereas it might have been desirable to collect some or all of those discarded cells.

[007] Previously, recovery problems were often overlooked, or subject to debate, with a view to the productivity of gross flow separation. Bovine sperm, for example, is relatively easy to collect and process and high purities in the beef and dairy industries may be desirable, even at the cost of disposing of as much as 90% of the sperm. However, this high productivity methodology is not acceptable for limited sperm. For example, a specific animal could have exceptionally desirable genetic qualities, but it could produce weak sperm samples for separation. A species could be rare, endangered, or difficult to collect, limiting the amount of sperm available for separation. A previously collected sample can be preserved, but the animal or species may no longer be available for subsequent collections. Regardless of the circumstances, the process of separating sperm with waste is undesirable in limited quantities or sperm with high value. There is therefore a need for a viable sperm separation method with improved efficacy in recovering sperm.

SUMMARY OF THE INVENTION [008] Certain embodiments of the claimed invention are summarized below. These embodiments are not intended to limit the scope of the claimed invention, but instead serve as brief descriptions of possible forms of the invention. The invention can encompass a variety of forms that differ from these summaries.

4/53 [009] One embodiment relates to an effective method of separating a sperm sample into a particle separation instrument. The method can begin with the step of establishing a coating fluid stream in the particle separation instrument and fluidizing a sperm sample in the coating fluid stream. The sperm can be guided with the particle separation instrument that can also differentiate viable X-chromosome carrying sperm and / or viable Y-chromosome carrying sperm from the rest of the sample. Viable X chromosome carrying sperm and / or viable Y chromosome carrying sperm can then be collected. One or more of the separation parameters measured on the particle separation instrument can be determined on the particle separation instrument. Minimum productivity and minimum purity can be established for separation and separation efficiency can be determined from the measured separation parameters determined while separating. One or more of the instrument's parameters can be adjusted to increase separation efficiency while maintaining minimum productivity and maintaining minimum purity.

[0101 Another embodiment relates to an effective method of separating a sperm sample in a particle separation instrument that can begin with the step of establishing a coating fluid stream in the particle separation instrument. A sperm sample can flow into the coating fluid stream. The sperm can be guided with the particle separation instrument and then differentiated from the rest of the sample as viable X-chromosome carrying sperm and / or viable Y-chromosome carrying sperm. The sperm

5/53 viable X chromosome and / or sperm carriers viable Y chromosome can then be collected. One or more of the measured separation parameters can be determined on the particle separation instrument. Minimum separation efficiency and minimum purity can be established. Productivity can be determined based on separation parameters measured during separation. One or more of the instrument's parameters can then be adjusted to increase productivity while maintaining minimum separation efficiency and minimum purity.

[011] Yet another embodiment relates to an effective method of separating a sperm sample. The method can begin with the steps of standardizing the concentration of a sperm sample and standardizing the pH of a sperm sample and can continue with staining the sperm sample with a single dilution to provide a sperm sample stained having a concentration between about 160 x 10 ⁶ sperm per microliter and about 640 x 10 ⁶ sperm per microliter. The sperm can be analyzed in a particle separation instrument that is operated in a way that stops the separation of any coincident events while reaching a purity of at least 90%. Viable X chromosome carrying sperm and / or viable Y chromosome carrying sperm can then be collected. Between about 25 percent and 50 percent of the sperm sample processed using the particle separation instrument can be separated or collected from an enriched population of X-chromosome-enriched sperm and / or a population of enriched Y-chromosome sperm. .

| 012] BRIEF DESCRIPTION OF THE DRAWINGS

6/53 [013 | FIG. 1 illustrates a flow cytometer scheme for sperm separation according to certain embodiments described here.

[0141 FIG. 2 illustrates a schematic of a microfluidic chip for sperm separation according to certain embodiments described here.

[015 | FIG. 3 illustrates a graphical representation of various separation parameters acquired in a flow cytometer while separating sperm according to the various embodiments described here.

[016] FIG. 4 illustrates a graphical representation of various separation parameters acquired in a flow cytometer while separating sperm according to the various embodiments described here.

[017 [FIG. 5 illustrates a graphical representation of various separation parameters acquired in a flow cytometer while separating sperm according to the various embodiments described here.

[018] FIG. 6 illustrates a flow chart of a method according to certain embodiments described here.

[019] FIG. 7 illustrates a flow chart of a method according to certain embodiments described here.

| 020] FIG. 8 illustrates a graphical representation of data produced according to the embodiments described here.

| 021 | FIG. 9 illustrates a graphical representation of data produced according to the embodiments described here.

[022] Although the present invention can be realized with various modifications and alternative forms, specific embodiments are illustrated in the figures and described here by means of illustrative examples. It should be understood that the detailed figures and descriptions are not intended to limit the scope of the invention to the particular disclosed form, but that all

7/53 the modifications, alternatives, and equivalents covered by the spirit and scope of the claims are intended to be encompassed.

MODES FOR CARRYING OUT THE INVENTION [023] As used here, the term instrument parameter should be understood as including definitions relating to the conditions of analysis and / or separation into, from, and relating to an instrument, where such definitions can be modified by manual or automatic adjustments to the instrument. In the case of a flow cytometer, or other similar instruments, instrument parameters can include sample pressure, sample flow, coating pressure, coating flow, drop frequency, drop amplitude, coincidence abort logic, intercept regions , separation logic, and other similar definitions.

[024] The term separation parameters can include those conditions relating to preformed separation in a particle separation instrument. Separation parameters can include separation parameters measured in addition to parameters that are determined offline, estimated by an operator, and conditions relating to a separate population of particles or cells.

[025] Measured separation parameters can include those conditions relating to directly measured, calculated, or determined separation in a particle separation instrument while analyzing and / or separating a population of particles or cells. In the case of a flow cytometer, or other similar instruments, the measured separation parameters can include: event rate; separation rate; separation effectiveness; interruption rate; percentage of death on entry; live oriented gate percentage ', reason applies in relation to peak; or the percentage of events in other separation gates, such as an Xsort gate or a Y-sort gate.

8/53 [026] As used here, the term coincidence event can be understood as a single event in a particle separation instrument where one or more particles or cells are too close to be separated for individual collection, and where only one of the two cells or particles is desirable for collection. In the case of a droplet-jet air flow cytometer, a coincident event may occur when two sperm are close enough that they will end up in the same droplet but only one of these two cells is desired for collection. In a microfluidic chip or fluid change separator, a coincident event can occur when two particles or cells are so close that any mechanism to change the particle's trajectory will deflect both particles together, when only one of the particles is desirable for collection .

[027] The term separation effectiveness can be understood to refer to the recovery of particles or cells in terms of the percentage of particles or cells separated or collected from a group of cells or particles that are analyzed. The group of cells analyzed can be the total number of cells analyzed or it can be a subset of the total number of cells analyzed, such as the cells analyzed determined to be viable or otherwise desirable for analysis and potential collection.

[028] With regard to separation, the term productivity, as used here, can be understood to refer to the number of particles or cells separated or collected per unit time.

[029] With regard to separation, the term purity can refer to an actual or estimated percentage of cells or particles in the population of particles or cells collected or separated having the characteristic for which the particles were separated. In the case of sperm, purity can refer to the percentage of

9/53 X-chromosome-bearing sperm in a separate population for X-chromosome-bearing sperm or the percentage of Y-chromosome-carrying sperm in a separate population for Y-chromosome-bearing sperm regardless of the viability of the separate spermatozoa.

[030] Certain aspects disclosed here relate to an effective method of separating a sperm sample into a particle separation instrument. Particle separation instruments can include air flow jet cytometers, such as the MoFlo SX, MoFlo XDP (Beckman Coulter, Miami FL, USA); however, other commercially available flow cytometers could be modified for sperm separation as well. Air flow cytometers can be equipped with guiding features such as guiding nozzles for sperm orientation, optics for uniform cell illumination, and / or radially uniform optics for collecting fluorescence emissions from all cells regardless of their orientation . Flow cytometers having different flow chambers may also be used, such as closed chamber flow cytometers, or cuvettes. In addition, devices such as microfluidic chips with separation functions can be used according to certain embodiments described here.

[031] Some embodiments described here relate to screening and / or optimizing the effectiveness of separation, while other embodiments described here relate to tracking the effectiveness of separation and maintaining at least a minimum threshold of separation effectiveness . Both embodiments introduce the new measured parameter of separation efficiency in particle separation instruments. In the case of sperm, the separation efficiency can be the percentage of sperm collected in a separate group in

10/53 compared to the total number of sperm analyzed, or the percentage of sperm collected compared to the total number of sperm analyzed determined to be viable sperm. When the efficacy of separation is seen in terms of the percentage of sperm collected in relation to the total population of sperm analyzed, dead sperm, or sperm characterized as having compromised or non-viable membranes, can contribute to significant losses in the efficiency of separation.

[032] One embodiment described here provides a synergistic combination including staining methodologies that can reduce the numbers of dead sperm and which, in some cases, can improve the ability of flow cytometers to differentiate X-chromosome-bearing and sperm-carrying sperm. Y chromosomes. Such a synergistic combination provides a methodology for drastically reducing the amount of discarded sperm of a desired sex that may have previously been discarded as dead or not targeted. Yet another beneficial aspect of the improved staining methodology provides sperm at higher concentrations for separation than two-step staining procedures. As will be further described below, higher sperm concentrations can provide good event rates for acceptable productivity even when operating at high purities and low sample fluid flow rates, which can further improve sperm alignment and orientation.

[033] Certain aspects of this disclosure provide methods for improving the effectiveness with which a sample is separated, while operating in a manner in which all coincident events are rejected. Previous methodologies may have suggested recovery can be improved

11/53 by operating flow cytometers in a mode that accepts all coincident events. A coincident event can be understood as a particle detected in a flow cytometer that cannot be separated from an undesirable particle, where the unwanted particle can be a particle of the wrong sex, a dead particle, an unguided particle, or a non-oriented particle. otherwise identifiable that would not be collected. A common example would be the case of a desirable particle and an undesirable particle being placed within the same droplet. Most flow cytometers intercept such particles in the interest of preserving the purity of the separate sperm sample.

Obtaining and coloring sperm for separation [034] A population of sperm can be obtained in the form of pure semen, extended sperm, frozen-thawed sperm or combinations thereof. The sperm population can be obtained from the same location where the remaining steps are performed, or it can be extended in a sperm buffer suitable for transport to a separation facility. Once obtained, the sperm can be kept at room temperature, refrigerated, or even frozen in an appropriate buffer for later use. Sperm for staining and separation can be purchased from a mammal, or storage sperm can be purchased, such as a frozen or refrigerated reed obtained from storage. Alternatively, frozen or extended sperm can be pooled.

[035] The sperm population may originate from mammals, such as a non-human mammal listed by Wilson, D.E. and Reeder, D.M., Mammal Species of the World, Smithsonian Institution Press, (1993), the entire contents of which are hereby incorporated by reference.

12/53 [036] When collecting, or thawing, or even grouping, sperm can be checked for concentration, pH, motility, and / or morphology. In addition, antibiotics can be added before additional processing steps.

[037 | Once obtained, the sperm can optionally be standardized to a predetermined concentration and / or towards a predetermined pH. Each of the predetermined concentration and pH can be specific to different species, or even different breeds of animals within a species. In one embodiment, the sperm can be combined with an initial buffer in the form of a high capacity buffer. Exemplary buffers may include TRIS citrate, sodium citrate, sodium bicarbonate, HEPES, TRIS, TEST, MOPS, KMT, TALP, and combinations thereof. Any buffer having a high pH buffering capacity can also be employed, and can be used in combination with additional components that promote sperm viability such as egg yolk, and sources of citrates or citric acid. In addition, antioxidants and antibiotics can be used in the initial buffer to promote sperm viability.

| 038] The initial buffer may be set at a predetermined pH to normalize the pH of all sperm samples obtained. In one embodiment, the buffer is adjusted to a pH of 7.2. In addition, semen can become increasingly acidic over time, possibly due to proteins in the seminal fluid, or due to acidic by-products of dead or dying cells. The initial buffer introduces enough free proton binding bonds (ie, H ⁺ ) to keep the pH close to the predetermined target. Even in light of the natural tendency of the sperm to become more acidic over time, the initial / 53 buffer provides a means for stabilizing the pH throughout additional processing steps.

[039] As an example, the sperm sample can be diluted in the high capacity buffer in ratios from about 1: 1 to about 1:10. The resulting mixture will have a sperm concentration many times below the natural sperm concentrations for a particular species. The extended sperm can be centrifuged in order to re-concentrate the sperm. The centrifugation of the sperm and removal of the supernatant allows the sperm to be re-concentrated to a predetermined concentration. The predetermined concentration can be selected based on additional sperm processing steps. For example, in the case of cattle separated by sex, sperm can be reconcentrated to between about 2400 million sperm per ml and about 900 million sperm per ml to simulate a natural range of concentrations. Other concentrations, such as between about 1400 million spermatozoa per ml and about 2100 million spermatozoa per ml, or between about 1700 million spermatozoa per ml and about 2100 million spermatozoa per ml, can also be reached for processing additional.

[040 | Adjusting the concentration and pH of the sperm can provide a uniform starting point for further processing. For example, a relatively consistent pH and concentration can provide greater predictability in sperm staining, for example with a DNA-selective dye. If each sample is adjusted to the same predetermined pH and concentration, fewer tests may be required for each new collection to ensure adequate color for gender-based separation.

| 041 | The sperm population will include X-chromosome-bearing sperm and sperm-carrying sperm.

14/53 Y chromosome. Additionally, each of the X chromosome carrying sperm and the Y chromosome carrying sperm will include viable sperm and non-viable sperm. Viable spermatozoa can be considered spermatozoa with intact membranes while non-viable spermatozoa can be considered spermatozoa with compromised membranes. The distinction between viable sperm and non-viable sperm in conventional sperm separation is determined with the inclusion of an extinction dye that permeates sperm with compromised membranes. Sperm that tend to be dead or dying absorb the extinction dye and produce fluorescence signals distinct from the remaining sperm population, while sperm having intact membranes tend to be viable sperm that will prevent extinction dye uptake. Viable sperm, at the appropriate dosage, will generally be able to achieve fertilization in artificial insemination, while non-viable sperm, or sperm with compromised membranes, may be unable to achieve fertilization in artificial insemination or will have a greatly reduced ability to do so. . However, some fertilizing-capable sperm may have compromised membranes, and some sperm with intact membranes may be unable to fertilize.

[042] Whether standardized or not, sperm can be stained with a staining buffer to introduce a selective DNA dye. In the staining step, at least a portion of the sperm population is incubated with a staining buffer and a DNA-selective fluorescent dye in order to stoichiometrically stain the DNA content of each cell in the cell population. Hoechst 33342 tends to be less toxic than other dyes

DNA selective 15/53. The vehicle for delivering this dye can be in the form of a modified TALP buffer adjusted to a pH of about 7.4. Hoechst 33342 is described in U.S. Patent 5,135,759 and is commonly used for this purpose. However, other UV excitable dyes, as well as visible light excitable dyes, fluorescent polyamides, fluorescent nucleotide sequences, and sex specific antibodies, could also be used.

[043] Sperm in a natural state are often not permeable to such dyes. In order to produce uniform staining, the first staining step may include incubating at least a portion of the sperm population at an elevated temperature in a staining buffer at an elevated pH in addition to the dye. Examples of suitable first staining buffers may be a TALP, TES-TRIS, TRIS citrate, sodium citrate, or an EIEPES-based medium, each described in W02005 / 095960, incorporated herein by reference. An exemplary modified TALP described in WO2001 / 37655, incorporated herein by reference, is illustrated in Table 1.

TABLE 1 - Modified TALP Buffer

Concentration ingredient

95.0 mM NaCI

3.0 mM KCI

NaHPO <0.3 mM

10.0 mM NaHCO ₃

0.4 mM MgCl ₂ 6H ₂ O

2.0 mM Na Pyruvate

Glucose 5.0 mM

25.0 mM Na Lactate

HEPES 40.0 mM bovine serum albumin 3.0 mg / mL

16/53 [044] As an example, the sperm population, or a portion of the sperm population, could be diluted with the first buffer to between 640 x 10 ⁶ and 40 x 10 ⁶ spermatozoa / mL, up to about 320 x 10 ⁶ and 80 x 10 ⁶ sperm / ml, or up to about 160 x 10 ⁶ sperm / ml in the first buffer. The DNA-selective fluorescent dye can be added to the sperm suspended in the first buffer at a concentration of between about 10 μΜ and 200 μΜ; between about 20 μΜ and 100 μΜ, or between about 30 μΜ and 70 μΜ. The pH of the first buffer can be between about 6.8 and 7.9; about 7.1 and 7.6; or about 7.4 in order to help ensure uniform staining of nuclear DNA. Those of ordinary skill in the art will appreciate that the pH can be raised with the addition of NaOH and lowered with the addition of HCl.

[045 | The sperm population can be incubated at 3039 ° C, between about 32-37 ° C, or at about 34 ° C. The incubation period can vary between about 20 minutes and about an hour and a half, between about 30 minutes and about 75 minutes, or for about 45 minutes to about 60 minutes. As an example, the sperm population can be incubated for about 45 minutes at 34 ° C. Even within a single species, the concentration and pH of sperm and other factors affecting staining ability can vary from animal to animal. Those with ordinary skill can appreciate minimal variations for sperm incubation between species and even between breeds or animals of the same breed to achieve uniform staining without over-staining a sperm population.

| 046] In addition to the DNA-selective fluorescent dye, an extinction dye can be applied for the purpose of permeating sperm with compromised membranes and extinguishing the signals they produce. A death extinction dye can be

17/53 understood to include dyes that differentially associate with sperm with compromised membranes. It may be that these dyes enter the sperm with compromised membranes more easily because the membranes are disintegrating or otherwise increasingly porous. It may also be that these extinction dyes readily enter all sperm and that healthy sperm act to actively pump the extinction dyes out faster than sperm with compromised membranes. In either case, the sperm with which the death extinction dyes are associated includes a large portion of the sperm dying and dying, although not necessarily all the sperm dead and dying. The extinct signals produced from sperm with compromised membranes having an association with the extinction dye are distinct enough from healthy sperm signals that they can be removed from the additional analysis and separation applied to viable sperm.

[047] In one embodiment, a second staining step is carried out which further reduces the concentration of the sperm and introduces the death extinction dye. The pH of the second staining solution can be directed to reach a target pH in the final sperm sample. Exemplary descriptions of two-step staining processes are described in Published International PCT Application WO 201 1/123166 and International PCT Application / US12 / 58008, the entire disclosure of both of which is incorporated herein by reference.

[048] In another embodiment, the extinction dye and the DNA selective dye are applied together in a single treatment. In this embodiment, the extinction dye is incubated together with the DNA-selective dye at a temperature

18/53 high in the modified TALP which may be at a pH of 7.4. In this embodiment it is believed that there is a synergy when the sperm is standardized at a high pH of about 7.2 before staining at 7.4. In this way, the pH to which the sperm is exposed remains in a constant range with minimal variations. Because both the staining buffer and the initial extender have high buffering capacities, it is believed that the natural tendency of sperm to become more acid over time will be avoided. Additionally, by minimizing the changes in pH seen by sperm, it is believed that sperm are in a healthier condition to cope with the various pressures and stresses borne by the sex-based separation process.

Stained Sperm Separation [049] Previously, particle separation instruments operated for the purpose of sperm separation were based on the principle of achieving high levels of productivity in terms of sperm separated per second. However, separation can be performed with high efficiency in such a machine in order to recover as much of the desired sperm as possible. Whereas previous focuses on productivity and / or purity fail to achieve significant effectiveness with an ejaculate. For example, a MoFlo XDP, available from Beckman Coulter (Miami FL, USA), can be set for event rates of around 40,000 events per second, to reach between about 4,000 and about 8,000 separations per second, while achieving 90 percent purity. However, higher productivity (separation rates) can be achieved at the expense of one or both of purity and effectiveness. In a synergistic combination with improved staining methods, higher sperm concentrations, and lower inlet deaths,

19/53 vehicle for improving separation rates while maintaining separation efficiency and improved standard purities.

[050] Whether standardized or not and stained in a single step or in two steps, the sperm population can be separated by a particle separation instrument, such as a flow cytometer. With reference to FIG. 1 illustrates an air jet flow cytometer (10), although the separation can be performed with microfluidic chips or other types of flow cytometers, including flow cytometers having closed chambers and cytometers incorporating ablation lasers. The flow cytometer (10) includes a cell source (12) for producing a sperm sample stream, such as a stained sperm sample stream, for separation. The rate at which the sperm sample is delivered to the nozzle (14) can be considered the sample flow rate, and can be determined by a sample pressure applied to the cell source (12). The flow of the colored sperm sample is deposited into a nozzle (14) and introduced into, or flows into, a fluid stream (16) of the coating fluid (18). The coating fluid (18) can be supplied by a source of coating fluid (20) such that the cell source (12) supplies the sperm to the coating fluid (18) are concurrently fed through the nozzle (14). The coating fluid (18) can be supplied at a rate of the coating fluid that is determined by a coating pressure applied to the source of the coating fluid (20). In this way, the coating fluid (18) forms a fluid stream coaxially surrounding the sample having colored sperm leaving the nozzle (14) in the nozzle orifice (22). By providing an oscillator (24) that can be precisely controlled with an oscillator control (26), pressure waves can be established inside the nozzle (14) and transmitted to fluids exiting the nozzle (14) in the nozzle orifice (22) . In response to pressure waves, the fluid stream (16) exiting the nozzle orifice (22) forms

20/53 eventually regular droplets (28) at precise intervals. The frequency, and to some extent the shape of the droplets formed, can be controlled by a drop frequency and drop amplitude provided to the oscillator (24) or to the oscillator controller (26).

1051] Each droplet, thus formed, retains the coating fluid and the sperm sample that previously formed a portion of the fluid stream (16). Because the stained sperm are surrounded by the fluid stream (16) or the coating fluid environment, the droplets (28) ideally contain individually isolated sperm. However, sample concentration, sample pressure, and other instrument parameters dictate the frequency with which multiple cells will regularly occupy a single droplet, as well as the percentage of droplets containing sperm.

[052] The flow cytometer (10) acts to separate droplets based on the characteristics of the sperm that are expected to be contained within the droplets. This can be achieved through a cell sensor system (30) in communication with an analyzer (36). The cell sensor system (30) includes at least one sensor (32) responsive to the cells contained within the fluid stream (16). The cell sensor system (30) provides data to the analyzer (36), which can cause an action depending on the relative presence or relative absence of a characteristic of the cells in the fluid stream (16). Certain characteristics, such as the relative DNA content of sperm, can be detected by excitation with an electromagnetic radiation source (34), such as a laser generating an irradiation beam to which stained sperm are responsive. The source of electromagnetic radiation (34) can be a laser operated at UV wavelength, such as at about 355 nm. An example of such a laser may be a Vanguard 350 (available from Spectra-Physics), which operates at 350 mW. Various optics can be used / 53 to shape the laser beam profile, split the beam into more than one chain, or reduce the beam's power to one chain. Non-limiting examples of such optics can be found in WO / 2004/104178 and WO / 2001/85913, each of which is incorporated herein by reference.

[053] The characteristics of individual sperm, particularly the presence of an X chromosome or Y chromosome, can be determined from the fluorescence produced in response to the source of electromagnetic radiation (34). In particular, the cell sensor system configurations (34) may be in communication with an analyzer to provide a variety of fluorescence in formation, such as the direct fluorescence of an event, the lateral fluorescence of an event, or the amount of dispersion associated with an event. The analyzer (36) can include written instructions for analyzing the signals produced by one or more sensors (32) in the cell sensor system (30). The DNA-selective fluorescent dye binds stoichiometrically to sperm DNA. Because X-chromosome-bearing sperm contains more DNA than Y-chromosome-bearing sperm, X-chromosome-bearing sperm can bind to a larger amount of DNA-selective fluorescent dye than Y-chromosome-bearing sperm. fluorescence emitted by the linked dye after excitation, it is possible to differentiate between sperm carrying X and sperm carrying Y. Distinctions, such as sperm that are viable or non-viable, can be further differentiated to sperm oriented and not oriented by the analyzer (36) accordingly with interception regions incorporating interception logic.

1054] In order to achieve separation and isolation based on stained sperm characteristics, the light emitted can be detected by the sensor (32) and the information fed to an analyzer (36) coupled to a

22/53 droplet carrier that differentially loads each droplet (28) based on the characteristics of the colored sperm contained within that droplet (28). In this way, the analyzer (36) acts to allow the electrostatic deflection plates (36) to deflect droplets (28) based on whether or not they contain the appropriate particle or cell.

[055] As a result, the flow cytometer (10) acts to separate stained spermatozoa by causing the droplets (28) containing spermatozoa to be directed to one or more collection containers (40). For example, when the analyzer differentiates sperm based on a sperm characteristic, the droplets dragging sperm carrying the X chromosome can be positively charged and thus deflect in one direction, while the droplets dragging sperm carrying the Y chromosome can be charged negatively and thus deflect to the other side, and the waste stream (that is, droplets that do not drag a particle or cell or drag unwanted or inseparable cells) can be left uncharged and is thus collected in an undeflected stream in a suction tube or similar. Alternatively, one of the sperm carrying the X chromosome or the sperm carrying the Y chromosome can be collected, while the other is discarded with waste.

[056] A controller (42) can form a portion of the analyzer (36) or it can be a component external to the analyzer (36). The illustrated controller (42) can also represent a collection of individual controllers. The controller (42) can receive signals or instructions from the analyzer (36) and in response can modify one or more parameters of the instrument, such as sample flow, sample pressure, coating flow, coating pressure, drop frequency, or range of fall and the like. The controller (42) can also provide a / 53 interface for operator inputs to manually adjust the sample flow, sample pressure, coating flow, coating pressure, drop frequency, or drop amplitude and the like. The analyzer (36) can include written instructions for modifying instrument parameters in response to measured separation parameters, or modifications to instrument parameters can be manually performed by an operator by adjusting various settings. Modifications to the instrument parameters can be carried out on the analyzer (36) such as by changing the separation logic, interception logic, separation regions, or interception regions and other specific parameters to make separation decisions on the analyzer. Additional modifications to the instrument parameters can be made by a controller (42), to control various components external to the analyzer, such as to control the sample pressure, sample flow, coating pressure, coating flow, drop frequency, and amplitude of fall.

| 057 | Turning now to FIG. 2, an alternative particle separation instrument is partially illustrated in the form of a microfluidic chip (60). The microfluidic chip (60) can include a sample inlet (62) for introducing a sample containing particles or cells into a fluid chamber (64) and through an inspection zone (66). The sample introduced through the sample inlet (62) can be isolated from interior channel walls and / or hydrodynamically focused with a coating fluid introduced through a coating inlet (68). The sample can be interrogated in the inspection zone (66) with a source of electromagnetic radiation (34), such as a laser, arc lamp, or other source of electromagnetic electricity. The resulting emitted or reflected light can be detected by a sensor (32) and analyzed with an analyzer (36), as described in FIG. 1. Each of the pressure

24/53 coating, sample pressure, coating flow, and sample flow on the microfluidic chip can be manipulated in a similar way to an air jet flow cytometer, by automatic adjustments performed by executing written instructions on the analyzer (36 ) or manual adjustments made by an operator.

[058] Once inspected, particles or cells in the fluid chamber (64) can be mechanically diverted from a first flow path (70) to a second flow path (72) with a separator (74), to change the fluid pressure or deviation of fluid flow. Particles or cells can also be allowed to continue to flow along the first flow path (70) for collection. The illustrated separator (74) comprises a membrane which, when concave, can divert the particles to the second flow path (72). Other mechanical or electromechanical shifting devices such as transducers and switches can also be used to divert the flow of particles.

[059] FIG. 3 illustrates a bivariate graph representative of lateral fluorescence and direct fluorescence of a jet cytometer in the stained air jet, which can be generated by an analyzer (36). The visual representation of data can be used by an operator to receive feedback relating to the sample undergoing separation and to graphically demonstrate certain aspects of the current separation logic. Rl, for example, can be seen as an interception region that can be applied to the flow cytometer separation logic. Additional numeric input can be provided on an analyzer display (36). Such numerical input can be in the form of measured separation parameters, such as an event rate, an interruption rate, separation rate, separation efficiency, or the percentage of particles in any region or gate. Rl is illustrated as a region that can be considered the live oriented region, because Rl's borders include two dense populations

25/53 cells that reflect a closely related sperm X chromosome population and closely related sperm Y chromosome population. R2 is a defined interception region around non-viable sperm, or sperm with compromised membranes whose fluorescence is extinguished by a death-extinction dye. Although a variety of separation logic can be employed, two strategies relating to IR and R2 could be a first step in a separation logic whereby all events being in IR are accepted for further processing or interception. Alternatively, all events outside R2 are accepted for further processing or interception.

[060] FIG. 4 illustrates a univariate graph in the form of a histogram that can be produced by the analyzer (36) and generated in a graphical presentation to an operator. The data illustrated in FIG. 4 can represent the number of peak or lateral fluorescence signal intensity peaks within a certain period. In the case of spermatozoa, the sperm carrying the X chromosome and the sperm carrying the Y chromosome tend to have peaks of intensity ranging between 2 and 5%, depending on the species, and this difference is reflected in the bimodal distribution of the peaks of intensity seen in FIG. 3. Because X-chromosome-bearing sperm and Y-chromosome-bearing sperm tend to have different fluorescence values, each peak represents either X-chromosome-carrying sperm or Y-chromosome-carrying sperm. Based on the separation logic applied within from an analyzer (36), the population of cells in the histogram can be only those cells that have been determined to be viable oriented cells, such as those being in IR in FIG. 3, or may represent cells that have not been determined to be dead or undesirable, such as all

26/53 events except those in R2, A variety of separation parameters can be derived from the information contained within this histogram. For example, the level of distinction between the two peaks can provide an indication of what a separate purity might look like. FIG. 4 further illustrates measurements of relative intensity at the lowest point between the two groups, which can be considered a V value and a second relative intensity at the peak or peaks of the histogram in P. A visual inspection of a histogram can give the operator an idea of how a flow cytometer is operating, but previously written computer instructions for determining a P value, a V value, and a ratio of V to P have not been implemented in flow cytometers. The peak-to-peak ratio can be determined as a separation parameter measured periodically during the course of the separation. The ratio applies to peak, although not necessarily completely determining separation purities, it can provide a means to quickly estimate the purity values, either by executing written instructions on the analyzer (36), or by visual inspection by an operator . Alternatively, the peak to valley ratio can provide a value that can be used in a similar way.

[0611 Turning to FIG. 5, a second bimodal graph can be generated by the analyzer (36) in response to signals acquired by the cell sensor system (30). The bimodal graph can represent a first axis illustrating the peak intensity value of a direct fluorescence signal or the peak intensity of lateral fluorescence signal. As FIG. 4, the data illustrated in FIG. 5 can be intercepted such that only events being in IR in FIG. 3 are included. Alternatively, in the case of sperm, all events that are not at the death gate R2 may also be displayed.

27/53 [062] R3 can represent an X-sort gate for collection of sperm carrying X chromosome. The term X-sort gate can be used interchangeably here with the term X-gate. With reference to FIG. 5 can demonstrate how changing the dimensions of the intercept regions can affect efficiency, purity, and productivity. If the R3 region were expanded it could be seen that every second more sperm would be separated as sperm carrying the X chromosome resulting in higher separation efficiency and higher productivity. However, the expansion of the gate or R3 region would begin to include events having an increased probability of being sperm carrying the Y chromosome. In order to increase the separate purity of sperm, the R3 region can be made smaller and / or moved away. of the Y chromosome region. As fewer events occur within the X-sort gate, fewer sperm are separated in the population of sperm carrying the X chromosome and those that are are more likely to be sperm carrying the X chromosome, meaning that the collected purity can be increased. However, both the efficiency, in terms of collected cells, and the productivity, in terms of separations per second, will decrease as fewer events occur in the R3 region. In addition, as other parameters of the instrument are modified, the graphs illustrated in FIG. 3, FIG. 4, and FIG. 5 can change in shape and nature. For example, increasing the pressure of a sample or the flow rate of a sample can result in a reduction in the valley to peak ratio, or it can otherwise blur the bimodal distinction between X-chromosome-bearing sperm and Y-chromosome-bearing sperm.

[063] Turning to FIG. 6 illustrates a method (200) of effective sperm separation in the form of a flow chart. The method can

28/53 starts with the step of defining a purity (210), which can be a minimum threshold purity. The minimum purity threshold can be set by an operator based on the expected performance of a particle separation instrument as well as based on the expected performance of a particular ejaculate, or a particular animal. Alternatively, a minimum purity threshold can be established after a sample has been partially analyzed or separated. The minimum purity threshold can be placed on the analyzer (36) for comparison against various measured separation parameters, or can be maintained by an operator, to make manual adjustment to the particle separation device based on measured separation parameters. The minimum purity threshold can be set at about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98%, or about 99%.

1064] Productivity can be defined (220) before purity is defined, after purity is defined, or at the same time. Productivity can be determined in terms of separations per second or it can be defined as a minimum productivity threshold. It should be appreciated that sperm samples that are stained in a way that reduces the number of dead sperm and are separated at increasing concentrations can be separated at particularly high productivity. Additional increases in productivity can be achieved by expanding the separation regions and reducing the minimum purity threshold.

[065] The minimum productivity threshold can be set at about 3,000 separations per second, 3,500 separations per second, about 4,000 separations per second, about 4,500 separations per second, about 5,000 separations per second, about 5,500 separations per second, about 6,000 separations per second, about 6,500 separations

29/53 per second, about 7,000 separations per second, about 7,500 separations per second, about 8,000 separations per second, about 8,500 separations per second, about 9,000 separations per second, about 9,500 separations per second, about 10,000 separations per second, about 10,500 separations per second, about 11,000 separations per second, about 11,500 separations per second, about 12,000 separations per second, about 12,500 separations per second, about 13,000 separations per second, about 13,500 separations per second, or about 14,000 separations per second.

[066 | As soon as each of the minimum thresholds for purity and productivity are defined, a particle separation instrument can start, or continue, the particle analysis and separation operation (230). In the course of the operation, the separation parameters can be determined (240). The separation parameters can include those conditions relating to preformed separation in a particle separation instrument. Separation parameters can include measured separation parameters, parameters that are determined offline, parameters estimated by an operator, and conditions relating to a separate population of particles or cells. The measured separation parameters can be determined in the analyzer (36) and can include those conditions relating to directly measured, calculated, or determined separation in a particle separation instrument while analyzing and / or separating a population of particles or cells, such as the event rate, separation rate, separation effectiveness, interruption rate, percentage of death on entry, live oriented gate percentage, peak to peak ratio, or the percentage of events in other separation gates, such as an Xsort gate or a Y-sort gate.

30/53 [067] A purity for comparison with the minimum purity threshold can be estimated by an operator based on the graphical representations generated by the analyzer, as illustrated in FIG. 3, FIG. 4, and FIG. 5. A purity can also be determined offline, as in a subsequent purity analysis of sperm nuclei. Purity can also be estimated by executing instructions written on the analyzer (36). The analyzer (36) can evaluate the measured separation parameters, such as the ratio of peak to peak, to estimate purity. An algorithm for estimating purity can incorporate empirical data based on valid ratios in relation to previous peak coordinates with subsequently determined purities of sonicated sperm (sperm without flow or sperm nuclei).

| 068] The productivity determined in the analyzer (36) can be compared from the separation parameters measured directly against the minimum productivity threshold (260). In case both purity and productivity, determined in any case, are above their respective minimum threshold values, one or more parameters of the instrument can be adjusted to increase the separation efficiency (280). Instrument parameters can be adjusted manually by an operator, or the analyzer can execute written instructions automatically for varying sample pressure, sample flow, or one or more separation regions. As an example, where purities are determined to be well above the minimum purity threshold.

[069] As an example, the separation logic can be adjusted. The separation logic can be considered the logic applied by the analyzer (36) to determine which cells are separated and which are discarded with waste. The separation logic can include an interception logic that determines when coincident events will be intercepted

31/53 in the course of the separation. For example, when high purity is desired, all coincident events can be intercepted, whereas when high productivity is desired, an interception logic that accepts coincident events can be applied. Depending on the frequency and accuracy with which purity is determined, a percentage of coincident events can also be accepted.

[070] As another example, the separation gates or separation regions can be modified. When both purity and productivity are above their respective thresholds, the separation gates, such as the life gate illustrated in FIG. 3 like Rl, can be extended to include more events. Similarly, the X-sort gate illustrated in FIG. 5 like R3, the Y-sort gate illustrated in FIG. 5 such as R4, or both, can be extended to separate more particles.

1071] In one embodiment, a change in the drop frequency can reduce the number of coincident events by producing more droplets in a given period of time and with less droplets having more than one cell. Similarly, the fall amplitude can be modified.

| 072 | In one embodiment, the sample flow rate can be modified when the minimum purity and minimum productivity threshold are met. In order to increase the efficiency of the separation, the sample pressure, or correspondingly the sample flow, can be reduced. Such a reduction in sample flow increases efficiency by reducing the number of coincident events and improving cell alignment and orientation. Accordingly, in order to further increase efficiency, the separation regions can be expanded while reducing sample pressure or sample flow.

1073] The fluid flow in combination with the concentration of cells in the sample together directly affect the measured parameter of the

32/53 event rate. The measured parameter of the event rate can then be directed to improve the effectiveness of the separation. The event rate can be targeted between 2,000 and 20,000 events per second at standard sperm concentrations, such as a sperm sample between 75 and 100 million sperm per ml. At high sperm concentrations, such as 150 million sperm per mL and more, event rates can be targeted between 2,000 events per second and 35,000 events per second, or higher.

1074] In case any of the purity and productivity, determined in any case, are above their respective minimum threshold values, one or more parameters of the instrument can be adjusted to decrease the effectiveness of the separation, or to increase any of the purity or productivity (270). Instrument parameters can be adjusted manually by an operator, or the analyzer can execute written instructions automatically for varying sample pressure, sample flow, or one or more separation regions.

[075 | As an exemplary embodiment, when the minimum productivity threshold is exceeded, but the minimum purity threshold is not exceeded, the sample flow can be reduced, or one or more of the live oriented separation region (RI) or X-sort gate (R3) or Y-sort gate (R4) can be decreased to include fewer events, including those events that tend to be outside the required purity. Similarly, in the event that the interception logic has been operating in a coincidence acceptance mode, it can be switched to a coincidence rejection mode, or to a mode that rejects an increased percentage of coincident events. If the minimum purity threshold is met, but the minimum production threshold is not met, one or more separation regions can be increased in size to include more events.

33/53 [076] After any modifications, the particle separation instrument can continue to operate and the separation parameters can continue to be determined. Adjustments can then proceed to incrementally improve or maximize the effectiveness of the separation. Optionally, incremental adjustments towards maximum separation efficiency can stop as soon as purity or productivity approaches a predetermined range from their respective minimum thresholds.

[077] With reference to FIG. 7 illustrates a method (300) of effective sperm separation, while maximizing productivity, in the form of a flow chart. The method can start with the step of defining a purity (310), which can be a minimum threshold purity. The minimum purity threshold can be set by an operator based on the expected performance of a particle separation instrument as well as based on the expected performance of a particular ejaculate, or less a particular animal. Alternatively, a minimum purity threshold can be established after a sample has been partially analyzed or separated. The minimum purity threshold can be placed on the analyzer for comparison against various measured separation parameters, or can be maintained by an operator, to make manual adjustment to the particle separation device based on measured separation parameters. The minimum purity threshold can be set at about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98% or about 99%.

[078] A separation efficiency can be defined (320) before the purity is defined, after the purity is defined, or at the same time. The effectiveness of the separation can be determined in terms of the percentage of

34/53 spermatozoa separated or collected over a period of time in relation to the total sperm population analyzed during that period of time. The effectiveness of the separation can also be determined in terms of a yield in living cells. For example, the effectiveness of the separation can be determined as the percentage of cells separated or collected over a period of time in relation to the population of cells not considered to be dead or not viable (ie, all cells outside the R2 region seen in Figure 3).

[079] As soon as each of the minimum thresholds for purity and separation efficiency are defined, a particle separation instrument can start, or continue, the particle analysis and separation operation (330). In the course of the operation, the separation parameters can be determined (340). The separation parameters can include those conditions relating to preformed separation in a particle separation instrument. Separation parameters can include separation parameters measured in addition to parameters that are determined offline, estimated by an operator, and conditions relating to a separate population of particles or cells. The measured separation parameters can be determined in the analyzer (36) and can include those conditions relating to directly measured, calculated or determined separation in a particle separation instrument while analyzing and / or separating a population of particles or cells, such as such as the event rate, separation rate, separation effectiveness, interruption rate, percentage of death on entry, live oriented gate percentage, peak to peak ratio, or the percentage of events in other separation gates, such as a X-sort gate or a Y-sort gate.

[0801 A purity for comparison with the minimum purity threshold (350) can be estimated by an operator based on representations

35/53 graphics generated by the analyzer, as illustrated in FIG. 3, FIG. 4, and FIG. 5. A purity can also be determined offline, as in a subsequent purity analysis of sperm nuclei. Purity can also be estimated by executing instructions written on the analyzer (36). The analyzer (36) can evaluate the measured separation parameters, such as the ratio of peak to peak, to estimate purity. An algorithm for estimating purity can be developed from empirical data based on valid ratios in relation to previous peak coordinates with subsequently determined purities of sonicated sperm (eg, sperm without flow or sperm nuclei).

[081] The separation efficiency determined on the analyzer (36) can be compared from the separation parameters measured directly against the minimum separation efficiency threshold (360). In case both the purity and efficiency of the separation, determined in any case, are above their respective minimum threshold values, one or more parameters of the instrument can be adjusted to increase productivity (380). Instrument parameters can be adjusted manually by an operator, or the analyzer can execute written instructions automatically for varying sample pressure, sample flow, or one or more separation regions.

[082] As an example, the separation logic can be adjusted to increase productivity. The separation logic can be considered the logic applied by the analyzer (36) to determine which cells are separated and which are discarded with waste. The separation logic can include an interception logic that determines when coincident events will be intercepted in the course of the separation. For example, when high purity is desired, all coincident events can be intercepted, whereas when high separation productivity is desired, an interception logic that accepts coincident events can be applied. Alternatively, a percentage of coincident events can also be accepted.

| 083] As another example, the separation gates or separation regions can be modified. When both the purity and effectiveness of the separation are above their respective thresholds, the separation gates, such as the life gate illustrated in FIG. 3 like Rl, can be extended to include more events to increase productivity. Similarly, the X-sort gate illustrated in FIG. 5 like R3, the Y-sort gate illustrated in FIG. 5 such as R4, or both, can be extended to separate more particles.

[084] In one embodiment, a change in the drop frequency can reduce the number of coincident events by producing more droplets in a given period of time and with less droplets having more than one cell. Similarly, the fall amplitude can be modified.

[085] In one embodiment, the sample flow rate can be modified when the minimum purity threshold and minimum separation efficiency thresholds are met. In order to increase productivity, the sample pressure, or correspondingly the sample flow, can be increased. Such an increase in the sample flow increases the number of events per unit time, possibly to the detriment of efficacy and a slight detriment to purity. In order to further increase the productivity and efficiency of the separation, albeit at the expense of purity, the separation regions can be expanded while increasing the sample pressure, or sample flow.

[0861 Fluid flow in combination with the concentration of cells in the sample directly affects the measured parameter of the event rate. The measured parameter of the event rate can then be

37/53 aimed at improving separation efficiency while maximizing productivity. The event rate can be targeted between 2,000 and 20,000 events per second at standard sperm concentrations, such as a sperm sample between 75 and 100 million sperm per mL. At high sperm concentrations, such as 150 million sperm per mL and more, event rates can be targeted between 2,000 events per second and 35,000 events per second, and higher.

[087] In case any of the purity and effectiveness of the separation, determined in any case, are above their respective minimum threshold values, one or more parameters of the instrument can be adjusted to decrease productivity, or to increase any of the purity or separation effectiveness (370). Instrument parameters can be adjusted manually by an operator, or the analyzer can execute written instructions automatically for varying sample pressure, sample flow, or one or more separation regions.

| 088] As an exemplary embodiment, when the minimum separation efficiency threshold is exceeded, but the minimum purity threshold is not exceeded, the sample flow rate can be reduced, or one or more of the live oriented separation region (IR) or the X-sort gate (R3) or Y-sort gate (R4) can be reduced in size or swapped to include fewer events, effectively excluding more events that tend to be outside the required purity. Similarly, in the event that the interception logic has been operating in a coincidence acceptance mode, it can be switched to a coincidence rejection mode, or to a mode that rejects an increased percentage of coincident events. If the minimum purity threshold is met, but the minimum separation effectiveness threshold is not, one or more separation regions can be

38/53 increased in size or changed to include more events, including more events that are more likely to meet the purity threshold.

[089] After any modifications, the particle separation instrument can continue to operate and the separation parameters can continue to be determined. Adjustments can then proceed to incrementally improve or maximize productivity. Optionally, incremental adjustments in the direction of maximum productivity can stop as soon as the purity or effectiveness of the separation approaches a predetermined margin of its respective minimum thresholds.

| 090 | Various modifications to the method described in FIG. 6 and FIG. 7 can be implemented to accommodate different animals. In the case of cattle, a young genomic breeder may have a lower sperm count compared to more mature animals. The minimum purity threshold and / or productivity threshold can be adjusted accordingly to achieve an effective use of the sperm.

[091] Example 1 - standardization of sperm samples and staining in one step [092] Collection - Sperm were collected from five different bulls in a routine collection program using an artificial vagina. Each bull was collected two or three times in one day. Of the five bulls, two were Jersey bulls and three were Holstein bulls. All ejaculates contained more than 60% progressive motility and the sperm concentration varied from 857 million sperm per ml. to 2480 million sperm per ml. The ejaculates collected from the same bull were grouped, then divided into nine sperm samples for collection and staining treatments.

[093] Coloring - Portions of each bull ejaculate were stained with nine different methods.

39/53 [094] (A) Control (no standardization, separation in two steps) - A control was established that did not include the standardization step of collected ejaculates and in which the sperm was stained in two steps. Prior to staining, sperm samples were concentrated to between 1700 million sperm per ml and 1800 million sperm per ml by centrifugation or by adding an egg yolk buffer having a pH of 6.8, depending on the concentration initial sample.

[095] The sperm in the control group was diluted to 160 x 10 ⁶ sperm per ml in a modified TALP buffer, as described in Table 6, at a pH of 7.4. Each sperm sample in the control group was then incubated with 16-17 pL of Hoechst 33342 per ml (64-68 μΜ) of sample for 45 minutes at 34 ° C. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80 x 10 sperm per ml. The second modified TALP includes the components described in Table 1 with the addition of 4% egg yolk, yellow food coloring No. 6 at 50 μΜ (20 g / L) and the pH was lowered to 5.5 with the addition of HC1.

[096] (B) Extended (no standardization, two-step staining) - In the second group, the sperm was not standardized, but was extended with a buffer and 20% egg yolk. The sperm was then concentrated to between 1700 million sperm per ml and 1800 million sperm per ml in the same manner as described for group (A). The sperm was then diluted to 160 x 10 sperm per ml in a modified TALP buffer, and stained in the same way in two steps described in group (A).

[0971 (C) One Step I (no standardization, one step staining with 1% egg yolk) - In a third group, the sperm was collected and the concentration was adjusted in the same way as the group of

40/53 control (A). Each sperm sample was then diluted to 160 x 10 ⁶ sperm per ml in a modified TALP buffer at a pH of

7.4. The modified TALP buffer was substantially identical to the buffer described in Table 1, except that it included 1% egg yolk and yellow food coloring No. 6 at a concentration of 25 μΜ. Each sperm sample in this group was then incubated with 14-15 pL of Hoechst 33342 per ml (56-60 μΜ) for 45 minutes at 34 ° C. After incubation, the sperm remained at a concentration of 160 x 10 ⁶ sperm per ml.

10981 (D) Standardized I (standardized with egg yolk buffer a

%) - In this group, the sperm was standardized by adjusting the pH and sperm concentration before staining and separation. After collection, the sperm was diluted 1: 3 in an initial buffer having a pH of 7.2 as well as a high pH buffering capacity. The high capacity buffer was supplemented with 3% egg yolk. All samples were then centrifuged to decrease the sperm concentration to between 1700 million sperm and 1800 million sperm per ml. The standardized sperm was then stained according to the two-step method described in (A).

[099] (E) Standardized II (standardized with 10% egg yolk buffer, two-step staining) - In this group, the sperm was standardized by adjusting the pH and sperm concentration before staining in the same manner as described in group (D), except that the initial buffer was 10% egg yolk.

| 0100] (F) One Step and Standardized I (standardized with 3% egg yolk buffer, stained in one step with 1% egg yolk) - In this group, the sperm was standardized by adjusting the pH and concentration sperm before separation in the same way as described in group (D). The standardized / 53 sample was then stained with a one step staining process as described in group (C).

| 01011 (G) One Step and Standardized 11 (standardized with 10% egg yolk buffer, stained in one step with 1% egg yolk) - In this group, the sperm was standardized by adjusting the pH and concentration of the sperm before staining in the same manner as described in group (E). The standardized sample was then stained with a one-step staining process as described in group (C).

[0102] (H) One Step and Standardized III (standardized with 3% egg yolk buffer, stained in one step with no egg yolk) In this group, the sperm was standardized by adjusting the pH and sperm concentration before staining as described in group (D). The standardized sample was then stained with a one-step staining process as described in group (C), except that no one egg yolk was added to the one-step staining TALP.

[0103] (I) One Step and Standardized IV (standardized with 10% egg yolk buffer, staining in one step with no egg yolk) In this group, the sperm was standardized by adjusting the pH and sperm concentration before of separation in the same way as described in group (E). The standardized sample was then stained with a one-step staining process as described in group (C) except that no egg yolk was added to the one-step staining TALP.

[0104] Separation and data acquisition - Each of the stained samples was separated on a MoFlo SX (Beckman Coulter, USA). Those samples that were stained in a two-step process were separated at a concentration of 80 x 10 ⁶ sperm per ml, and those samples that were stained by a one-step process were separated at a concentration of 160 x 10 ⁶ sperm per ml. The data reported by the flow cytometer were recorded including

42/53 information relating to the rates of separation and interception of subpopulations of sperm. For example, the percentage of sperm intercepted as dead, as well as the percentages of sperm intercepted as live and overbanded, were recorded and averaged for the five bulls.

[0105] Results - A comparison of the percentage of spermatozoa that were oriented, not oriented and killed as determined by the separation parameters established in the flow cytometer is summarized in Table 2 below.

% of Oriented % of non-oriented % of dead Rate of Separation Overgrowth A) Control 58.29% 18.02% 16.89% 3500 4.32% B) Extended 60.54% 20.20% 8.71% 3400 10.36% C) Step I 61.04% 17.96% 12.31% 3500 5.65% D) Standardized 1 52.78% 18.14% 9.71% 2900 24.73% E) Standardized 11 55.20% 18.70% 6.04% 3200 23.44% F) One + Standardized Step 1 57.33% 20.35% 5.39% 3200 16.17% G) One Step + Standardized II 59.99% 18.89% 5.19% 3600 16.83% H) One Step + Standardized III 62.67% 22.02% 6.97% 3800 6.23% I) One Step + Standardized IV 63.49% 23.16% 5.61% 4100 5.38%

[0106] Compared to control (A), the One Step I (C), Standardized I (D), and Standardized II (E) groups each exhibited significantly lower dead populations with reductions of 4.58%, 7 , 18% and 10.85%, respectively. Based on these improvements, the steps of standardizing sperm samples before staining and modifying the staining process to a single step independently improve the sperm's ability to survive the separation process. Additionally, One Step and Standardized I (F), One Step and Standardized II (G), One Step and Standardized III (H), and One Step and Standardized IV (I) demonstrate a synergy by which the combined effect

43/53 standardization of an ejaculate and coloration of the ejaculate in a single step is greater than any of the improvements individually.

[0107] Regarding Table 2, it can be seen that Standardized 1 (D), Standardized II (E), One Step and Standardized I (F), and One Step and Standardized II (G) each seem to provide significant benefits in terms of reducing the number of dead sperm, but the percentage of targeted sperm has not improved. This may be related to the column indicated as overrange. Although more sperm were intercepted as alive for separation, there appears to be an increase in scattered signals above the separation gates. This signal can represent sperm that are stuck together or it can represent sperm that are attached to lipids in egg yolk. In either case, the general pattern emerges that greater amounts of egg yolk reduce the numbers of sperm, but may introduce a new issue and a balance may therefore be required.

| 0108] Example 2 - standardization of sperm samples and staining in one step [0109 | Collection - Sperm was collected from six different Jersey bulls in a routine collection program using an artificial vagina. All ejaculates contained more than 65% progressive motility and the sperm concentration ranged from 765 million sperm per mL to 1710 million sperm per mL Each sperm sample was divided into two parts in 15 mL tubes for two collections and staining treatments. PH measurements were taken at collection, and at each subsequent processing step.

[0110] Staining - Control (no standardization, separation in two steps) - A control was established that did not include the standardization step of collected ejaculates and in which the sperm was stained in

44/53 two steps. Prior to staining, sperm samples were concentrated to between 1700 million sperm per ml and 1800 million sperm per ml by centrifugation or by adding an egg yolk buffer having a pH of 6.8, depending on the concentration initial sample.

[0111] The sperm in the control group was diluted to 160 x 10 ⁶ sperm per ml in a modified TALP buffer, as described in Table 6, at a pH of 7.4. Each sperm sample in the control group was then incubated with 16-17 pL of Hoechst 33342 per ml (64-68 μΜ) of sample for 45 minutes at 34 ° C. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80 x 10 ⁶ spermatozoa per ml. The second modified TALP includes the components described in Table 1 with the addition of 4% egg yolk, red food coloring No. 40 at 50 pM (20 g / L) and the pH was lowered to 5.5 with the addition of HC1.

[0112] One Step and Standardized (standardized with 10% egg yolk, stained in one step with one percent egg yolk) - The sperm was standardized by adjusting the pH and sperm concentration before staining. After collection, the sperm was diluted 1: 3 in an initial buffer having a pH of 7.2 as well as a high pH buffering capacity. The high capacity buffer was supplemented with 3% egg yolk. All samples were then centrifuged to decrease the sperm concentration to between 1700 million sperm and 1800 million sperm per ml.

| 0113] The sperm samples were then diluted to 160 x 10 ⁶ sperm per ml in a modified TALP buffer at a pH of

7.4. The modified TALP buffer was substantially identical to the buffer described in Table 1, except that it included 1% egg yolk and yellow food coloring No. 6 at a concentration of 25 μΜ. Each

45/53 sperm sample in this group was then incubated with 16-17 pL of Hoechst 33342 per ml (64-68 μΜ) for 45 minutes at 34 ° C. After incubation, the spenna remained at a concentration of 160 x 10 ⁶ spermatozoa per ml.

| 0114 | Separation and data acquisition - Each sample was separated on a MoFlo SX (Beckman Coulter, USA). The control was separated at a concentration of 80 x 10 ⁶ sperm per ml, while the standardized sperm was separated at 160 x 10 ⁶ sperm per ml. The data were communicated by the flow cytometer and their average was then calculated for the 6 bulls.

[0115 | Results - FIG. 3 illustrates the recorded pH of the control (A) and the standardized ejaculate (B). These Values are reflected in TABLE 3 below. Although the standardized ejaculate is subject to an initial increase, a subsequent increase is avoided during staining and the following fall is also avoided. In addition, TABLE 4 illustrates similar benefits in reducing dead sperm that were seen in Example 1. Specifically, the standard sample that was stained in one step had 5.67% less dead sperm.

TABLE 3

Initial Before centrifugation After Centrifugation During the Coloring After staining Before cytometer Control (A) 6.34 6.34 6.25 7.22 7.07 6.59 Standardized (B) 6.34 7.12 6.85 7.18 6.98 6.98

TABLE 4

PV % in Oriented % of Dead Separation Rate Duplicates / T riplets Control 1.86 52.99 14.63 35.83 21.73 Standardized - One Step 1.97 57.22 8.96 37.00 24.59 Difference 0.11 4.23 -5.67 L17 2.86

46/53 [0116] Example 3 ~ standardization of sperm samples and staining in one step reduces dead sperm

10117] Collection - Sperm was collected from three different Jersey bulls and three different Holstein bulls in a routine collection program for a total of 17 collections. Each ejaculate was divided into two treatments.

| 0118] Staining - Control (no standardization, separation in two steps) - A control was established that did not include the standardization step of collected ejaculates and in which the sperm was stained in two steps. The sperm in the control group was diluted to 160 x 10 ⁶ sperm per ml in a modified TALP buffer, as described in Table 6, at a pH of 7.4. Each sperm sample in the control group was then incubated with 16-17 pL of Hoechst 33342 per ml (64-68 μΜ) of sample for 45 minutes at 34 ° C. After incubation, an equal volume of a second modified TALP was added, reducing the concentration to 80 x 10 ⁶ spermatozoa per ml. The second modified TALP includes the components described in Table 1 with the addition of 4% egg yolk, red food coloring No. 40 at 50 μΜ (20 g / L) and the pH was lowered to 5.5 with the addition of HC1.

(0119] Standardized III and One Step (standardized with 3% egg yolk buffer, one-step staining) - The remaining sperm was standardized by adjusting the pH and sperm concentration before staining and separation. was diluted 1: 3 in an initial buffer having a pH of 7.2 as well as a high pH buffering capacity. The high capacity buffer was supplemented with 3% egg yolk. The sperm sample was then diluted to 160 x 10 ⁶ sperm per ml in a modified TALP buffer at a pH of 7.4 The modified TALP buffer was substantially identical to the buffer described in Table 1, except that it included 1% egg yolk and yellow food coloring No. 6 at a concentration of 25 μΜ.

47/53 sperm sample in this group was then incubated with 14-15 pL of Hoechst 33342 per ml (56-60 μΜ) for 45 minutes at 34 ° C. After incubation, the sperm remained at a concentration of 160 x 10 ⁶ sperm per ml.

[0120] The control group was operated through a Legacy MoFlo SX (Beckman Coulter, Miami FL, USA) with a digital upgrade at a concentration of 80 x 10 ⁶ spermatozoa per mL, while the Standardized III and One Step was separated at a concentration of 160 x 10 ⁶ sperm per ml. Table 5 illustrates the percentage of cells at the death gate of each ejaculate and the mean. After separation, the percentages of sperm occurring in the death gates (R2 seen in FIG. 3) were indicated for both samples.

TABLES

Bull Death Gate (%) Ejaculate Number Bull CONTROL ONE STEP and STANDARDIZED III 01 Holstein bull 1 16% 12% 02 Holstein Bull 2 26% 6% 03 Jersey Bull 1 15% 7% 04 Holstein Bull 2 19% 3% 05 Jersey Bull 1 13% 6% 06 Holstein Bull 3 19% 12% 07 Bull Jersey 2 25% 14% 08 Holstein bull 1 25% 21% 09 Holstein Bull 2 20% 20% 10 Bull Jersey 3 9% 5% 11 Bull Jersey 2 19% 17% 12 Holstein Bull 3 15% 14% 13 Jersey Bull 1 10% 7% 14 Holstein bull 1 9% 6% 15 Holstein bull 1 9% 8% 16 Holstein Bull 3 17% 6% 17 Holstein Bull 3 16% 5% Average 17% 10%

[0121] Example 4 - Optimization of the efficiency of separation in flow cytometer

48/53 [0122] Sperm was collected from a Holstein bull and stained according to the protocol of Standardized III and One step described in the previous examples. The sample was placed in a Legacy MoFlo SX (Beckman Coulter, Miami FL, USA) with a digital upgrade. During separation, the coating fluid pressure was set at 40 PSI and the drop frequency was set to 64.9 KHz. The sample pressure was adjusted to target event rates of around 1500, 3500, 7500, 8500, 10,000 15000, 20000, 25000, and 30000.

[0123] The ejaculate in this example demonstrated a death gate of about 3% -5% that allowed large portions of sperm to be included in the live oriented gate ·, between 79.1% and 85.4%. The separation logic used in this separation focused on a live oriented sperm region (Rl). IR was established by an operator to retain a large portion of the sperm. The X-sort gate was similarly established by an operator with a target of 90% purity. The data were periodically digitally communicated to several samples at each event rate. The data for each event rate was averaged to provide averages for productivity (Separation Rate), separation effectiveness (Separation Rate / Event Rate), Vale ratio to Pico, interruption rate, as well as the percentage of the population of the Death gate (R2), the percentage of the population in the live oriented gate (Rl), the percentage of the sperm population in the X-Sort gate (R3), and the percentage of viable (live) sperm in the X-Sort gate. In addition, purities were determined offline for each sperm separated at each event rate setting. Purity was determined by sonicating the tails of 1 million sperm and collected at each event rate and measurement group in an offline purity analyzer. This measurement was performed twice for each group and the average was calculated.

49/53

TABLE 6

Vale / Pico (%) Event Rate (Hz) Separation Rate (Hz) Separation Fee! Event Rate (%) Rate of Interruption (Hz) Interruption Rate / Separation Rate Death Gale (%) LiveOriented (%) X-Sort Gale (%) X-Sort / Viable (%) X purity (%) 1 67.4% 1722 694 40.3% 48 7.0% 6.4% 82.9% 54.1% 57.7% 96.0% 2 66.6% 3697 1361 36.8% 141 10.4% 4.5% 84.9% 52.2% 54.6% 96.0% 3 63.4% 7377 2591 35.1% 414 16.0% 2.9% 85.4% 50.0% 51.5% 95.5% 4 63.4% 8515 3005 35.3% 522 17.4% 2.7% 84.9% 51.2% 52.6% 95.5% 5 62.1% 9891 3415 34.5% 645 18.9% 2.7% 84.4% 51.2% 52.6% 96.0% 6 54.7% 16686 4774 28.6% 1306 27.4% 2.8% 82.8% 47.1% 48.5% 93.0% 7 51.0% 19760 5080 25.7% 1604 31.6% 2.8% 81.8% 44.6% 45.9% 91.5% 8 47.5% 24839 5822 23.4% 2175 37.4% 2.8% 80.2% 43.5% 44.8% 90.0% 9 43.9% 29666 6332 21.3% 2706 42.7% 3.1% 79.1% 42.4% 43.7% 92.5%

[0124] Turning to FIG. 8 is shown a graphical representation of several measured separation parameters. In particular, it can be seen that low event rates reduce interception rates and improve the effectiveness of the separation. In particular, the interruption rate is 7% of the separation rate when the event rate is 1722.

[0125] Additionally, the synergistic effect of reducing dead sperm is illustrated by the fact that more than 50% of the sperm sample has been intercepted at the X-sort gate for event rates of less than 10,000 events per second. The low percentage of dead sperm in combination with the high percentage of live oriented sperm allows interception of an R3 region to be adjusted such that R3 invades the region of FIG. 5 where sperm are more likely to be sperm carrying Y chromosomes than sperm carrying X chromosome. Even when slightly invading this region, the purity checked after separation remained 96%, even though 54% of all sperm were included in the X-sort gate and 57% of all live sperm were included in the X-sort gate.

[0126 | The synergistic combination of improved staining techniques in combination with separation methods that focus on effectiveness can

50/53 can be seen as providing reliable sperm separation methods that can provide between 25% and about 40% yield in the total sperm population, and maintain purities greater than 90%.

[0127] Turning to F1G. 9 additional separation parameters are graphically illustrated from Table 6, including the purities for each group of event rates and the percentage of sperm in the live / oriented gate (Rl) and peak to valley ratio. Because a purity of 90% was targeted by an operator, trends in peak to valley ratio are not demonstrated in the measured purity but are reflected in the decreasing percentage of sperm in the X-Sort Gate.

[0128] One aspect of this disclosure designs more spatially effective flow cytometers, which can allow more separation heads in an available space, In such an arrangement, more flow cytometer separation heads can be dedicated to a single sperm sample, and each can be operated at improved efficiency, thereby combining the benefits of effective separation methods with high productivity.

] 0129] As can be easily understood from the above, the basic concepts of the present invention can be realized in a variety of ways. The invention involves numerous and varied embodiments of sex-separated sperm including, but not limited to, the best mode of the invention.

[0130] As such, the particular embodiments or elements of the invention disclosed by the description or shown in the figures or tables accompanying this application are not intended to be limiting, but rather exemplary of the numerous and varied embodiments generally encompassed by the invention or encompassing equivalents with respect to / 53 any particular element thereof. In addition, the specific description of a single embodiment or element of the invention may not explicitly describe all possible embodiments or elements; many alternatives are implicitly disclosed by the description and figures.

[0131] It should be understood that each element of a device or each step of a method can be described by a device term or method term. Such terms may be substituted where the implicitly broad coverage to which this invention is intended is made explicit. As just one example it should be understood that all steps in a method can be disclosed as an action, a means to take that action, or as an element that causes that action. Similarly, each element of a device can be disclosed as the physical element or the action that that physical element facilitates. As just one example, separator disclosure should be understood as encompassing disclosure of the act of separating - whether explicitly discussed or not - and, conversely, there was effectively disclosure of the act of separating, such disclosure should be understood as encompassing the disclosure of a separator and even a means for separation. Such alternative terms for each element or step are to be understood as being explicitly included in the description.

[0132] Additionally, for each term used it should be understood that, unless its use in this application is inconsistent with such an interpretation, common dictionary definitions should be understood to be included in the description for each term as contained in the Random House Webster's Unabridged Dictionary, second edition, each definition incorporated in this way by reference.

[0133] Furthermore, for the purposes of the present invention, the term one or an entity refers to one or more of that entity.

52/53

As such, the terms one or one, one or more and at least one (a) can be used interchangeably here.

[0134] All numeric values here are assumed to be modified by the term about, whether or not explicitly indicated. For the purposes of the present invention, the ranges can be expressed as from about one particular value to about another particular value. When such a range is expressed, another embodiment includes from one particular value to the other particular value. The recitation of numerical ranges by the endpoints includes all numerical values sub-summed within that range. A numeric range from one to five includes, for example, numeric values 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so on. It will be further understood that the end points of each band are significant in relation to the other end point, and independently of the other end point. When a value is expressed as an approximation by using the antecedent about, it must be understood that the particular value forms another embodiment.

] 0135] The background section of this patent application provides a statement of the area of effort to which the invention belongs. This section may also incorporate or contain paraphrasing of certain United States patents, patent applications, publications, or subject matter of the claimed invention useful in reporting information, problems, or concerns about the state of the technology to which the invention is directed. Any United States patent, patent application, publication, statement or other information cited or incorporated herein is not intended to be interpreted, understood or considered to be a prior art with respect to the invention.

[0136] The claims presented in this specification, if any, are hereby incorporated by reference as part of this description of the invention, and the applicant expressly reserves the right to

53/53 use all or a portion of such embedded content of such claims as an additional description to support any or all of the claims or any element or component thereof, and the claimant further expressly reserves the right to move any portion of or all the embedded content of such claims or any element or component of the description for the claims or vice versa as necessary to define the matter for which protection is sought by this application or by any subsequent application or continuation, division, or its application for continuation in part, or to obtain any benefit from, a reduction in fees under, or to comply with, the patent laws, rules, or regulations of any country or treaty, and such content incorporated by reference shall survive the entire term of this application including any subsequent continuation, division, or your request to continue in part or any of its reed tion or extension.

/ 5

Claims (18)

1. Effective method of separating a sperm sample in a particle separation instrument, characterized by comprising: a) the establishment of a coating fluid stream in the particle separation instrument; b) fluidizing a sperm sample into the coating fluid stream; c) the identification of viable X-chromosome carrying sperm and / or viable Y-chromosome carrying sperm in the sperm sample; d) the separation of viable X chromosome carrying sperm and / or viable Y chromosome carrying sperm from the rest of the sperm sample; e) the collection of viable X-chromosome carrying sperm and / or viable Y-chromosome carrying sperm; f) the determination of one or more separation parameters measured in the particle separation instrument, including the efficient selection of the analyzed cells, where the selection is calculated by continuously determining a ratio between a number of collected cells and a number of events representing the cells analyzed over a period of time; g) the establishment of a minimum productivity threshold of not less than 3,000 selections per second. h) the establishment of a minimum purity threshold of not less than 86% purity, and Petition 870190117208, of 11/13/2019, p. 9/13 2/5 i) the adjustment of one or more parameters of the instrument to increase the efficiency of the separation of analyzed cells while maintaining the productivity in terms of type per second, in terms of collections per second, above the minimum productivity threshold and purity above the threshold of minimum purity. 2. Method according to claim 1, characterized in that the measured separation parameters are selected from the group consisting of: event rate, separation rate, valley to peak ratios, interruption rate, percentage of particles in the death gate, percentage of particles in an X-sort gate, percentage of particles in a Y-sort gate, and percentage of particles in an oriented gate. Method according to claim 1, characterized in that the step of adjusting one or more parameters of the instrument comprises the adjustment of a separation logic. Method according to claim 1, characterized in that the step of adjusting one or more parameters of the instrument comprises adjusting a separation gate. Method according to claim 4, characterized in that the step of adjusting a separation gate comprises the step of modifying a separation region for collecting sperm carrying the X chromosome and / or a separation region for collecting sperm Y chromosome carriers to include more detected events representing analyzed cells. Method according to claim 1, characterized in that the particle separation device comprises a flow cytometer with jet in the air and the step of adjusting one or more parameters of the Petition 870190117208, of 11/13/2019, p. 10/13 3/5 instrument comprises adjusting a drop frequency or adjusting a drop amplitude. Method according to claim 1, characterized in that the step of adjusting the one or more parameters of the instrument comprises the reduction of a sample flow rate to increase and increase of a separation region. Method according to claim 1, characterized in that the ratio of sperm collected to the number of sperm in the separable sperm population is between 25% and 50%. Method according to claim 1, characterized in that the minimum purity threshold is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99%. Method according to claim 1, characterized in that the minimum productivity threshold is set at 3,000 separations per second, 3,500 separations per second, 4,000 separations per second, 4,500 separations per second, 5,000 separations per second, 5,500 separations per second, 6,000 separations per second, 6,500 separations per second, 7,000 separations per second, 7,500 separations per second, 8,000 separations per second, 8,500 separations per second, 9,000 separations per second, 9,500 separations per second, 10,000 separations per second, 10,500 separations per second, 11,000 separations per second, 11,500 separations per second, 12,000 separations per second, 12,500 separations per second, 13,000 separations per second, 13,500 separations per second, or 14,000 separations per second. Method according to claim 1, characterized in that the step of adjusting one or more parameters of the instrument to increase the separation efficiency while maintaining productivity Petition 870190117208, of 11/13/2019, p. 11/13 4/5 minimum and minimum purity is maintained additionally understand the steps of estimating a purity and comparing the estimated purity with the minimum purity. Method according to claim 11, characterized in that the step of estimating the purity further comprises the step of evaluating the valley to peak ratios determined in the particle separation instrument. Method according to claim 12, characterized in that the valley ratios to the peak are compared with empirical data to predict current purity. Method according to claim 13, characterized in that the step of comparing the valley ratios in relation to the peak with empirical data is performed by executing written instructions stored in a processor associated with the particle separation instrument. Method according to claim 1, characterized in that it further comprises the step of adjusting a sperm concentration in the sperm sample to optimize the separation efficiency. Method according to claim 1, characterized in that the step of adjusting one or more parameters of the instrument to increase the efficiency of the separation while maintaining the minimum productivity and maintaining the minimum purity additionally comprise the adjustment of one or more parameters of the instrument to maximize separation efficiency while maintaining minimum productivity and maintaining minimum purity. Method according to claim 1, characterized in that the sperm sample is obtained from a mammal having a low sperm production. Petition 870190117208, of 11/13/2019, p. 12/13 5/5 Method according to claim 17, characterized in that the mammal comprises a young genomic breeder. Petition 870190117208, of 11/13/2019, p. 13/13 1/7